Tamoxifen Monotherapy Research Articles

Abstract A1-06: Recurrent mutations of hormone-positive breast cancer and association with outcome

Abstract Background: Relationships between recurrent somatic mutations and outcome in estrogen receptor positive (ER+) breast cancer have not been extensively studied as the original discovery efforts were from either heterogeneously treated patients or follow up was too brief. Targeted massively parallel sequencing (MPS) analysis was therefore conducted on DNA extracted from archived formalin-fixed breast primaries from a cohort of over 600 patents from British Columbia treated with five years of adjuvant tamoxifen monotherapy and followed for over 10 years (Nielsen et al CCR 16:5222, 2010). Methods: Genes were selected for targeted sequencing by meta-analysis of five large-scale breast cancer sequencing studies and manual review of breast cancer literature. In total, 83 genes were identified and 3286 probes were designed to tile across all known exons. Minimum starting input DNA was 50ng (mean=189.1ng). Illumina sequencing libraries were constructed, indexed, pooled, and enriched for target sequences by hybrid-capture followed by paired-end 100bp reads. The Genome Modeling System was used to perform single-tumor somatic variant prediction. Variant calls were filtered to include only targeted regions and exclude variants with global minor allele frequencies greater than 0.1% in unmatched non-tumor samples from 1000 genomes, NHLBI exome, and TCGA datasets. Kaplan-Meier univariate and multivariate survival analyses (including mutation status, clinical features and intrinsic subtype by qPCR) were performed for breast-cancer-specific and relapse free survival. Results: A total of 625 samples met minimum quality controls of 80% targeted space covered at 20X or greater. On average, each sample had 332M aligned bases and a mean coverage of 134.3X. In total, 3,628 variants were identified including 2,066 missense, 188 nonsense, and 298 frame shift insertions or deletions. Novel hot spots for recurrent mutation were identified in several genes including a splice site mutation in CBFB. Results indicate significant associations between mutation status and improved survival for MAP3K1, ERBB3, ARID1B, PIK3CA and SMG1 or worse survival for DDR1, NF1, FOXC1 and TP53. Six Y537S/C, two E380Q and 5 potentially novel ligand-binding-domain mutations were identified in ESR1. Such mutations were recently reported to be associated with resistance to hormone therapy but were discovered here in as many as 2.1% of pre-treatment samples. Analysis will be presented regarding the use of relapse events to differentiate passenger from driver events. Conclusion: Multiple recurrently mutated genes have both positive and negative associations with prognosis in tamoxifen monotherapy treated breast cancer populations. Associations with poor outcome suggest that DDR1, NF1 and FOXC1 are high priorities for pharmacological interventions. Citation Format: Obi L. Griffith, Malachi Griffith, Nicholas C. Spies, Jingqin Luo, Jasreet Hundal, Christopher A. Miller, David E. Larson, Robert Fulton, Shuzhen Liu, Samuel Leung, Richard K. Wilson, Torsten O. Nielsen, Elaine R. Mardis, Matthew J. Ellis. Recurrent mutations of hormone-positive breast cancer and association with outcome. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-06.

Differences in clinical importance of Bcl-2 in breast cancer according to hormone receptors status or adjuvant endocrine therapy

BackgroundBcl-2 plays an anti-apoptotic role, resulting in poor clinical outcome or resistance to therapy in most tumor types expressing Bcl-2. In breast cancer, however, Bcl-2 expression has been reported to be a favorable prognostic factor. The positive correlation of Bcl-2 with estrogen receptor (ER)/progesterone receptor (PR) status, and endocrine therapy frequently given for hormone receptor-positive tumors, may obscure the independent pathobiological role of Bcl-2. We constructed a large systematic study to determine whether Bcl-2 has an independent role in breast cancer.MethodsBcl-2 expression was immunohistochemically evaluated and compared with other clinicopathological factors, including clinical outcome, in 1081 breast cancer cases with long follow-up, separately analyzing 634 cases without any adjuvant therapy and 447 cases with tamoxifen monotherapy. The χ2-test for independence using a contingency table, the Kaplan-Meier method with the log-rank test, and a Cox proportional hazards model were used for the comparison of clinicopathological factors, assessment of clinical outcome, and multivariate analyses, respectively.ResultsIn both patient groups, Bcl-2 expression strongly correlated with positive ER/PR status, low grade, negative human epidermal growth factor receptor 2 (HER2) status, and small tumor size, as previously reported. Bcl-2 expression did not independently predict clinical outcome in patients with ER-positive and/or PR-positive tumors or in those who received tamoxifen treatment; however, it was an independent unfavorable prognostic factor in patients with ER-negative/PR-negative or triple-negative (ER-negative/PR-negative/HER2-negative) tumors who received no adjuvant therapy. The latter was even more evident in postmenopausal women: those with hormone receptor-negative or triple-negative tumors lacking Bcl-2 expression showed a favorable outcome.ConclusionBcl-2 expression is an independent poor prognostic factor in patients with hormone receptor-negative or triple-negative breast cancers, especially in the absence of adjuvant therapy, suggesting that the anti-apoptotic effect of Bcl-2 is clearly exhibited under such conditions. The prognostic value of Bcl-2 was more evident in postmenopausal women. The present findings also highlight Bcl-2 as a potential therapeutic target in breast cancers lacking conventional therapeutic targets such as triple-negative tumors. The favorable prognosis previously associated with Bcl-2-positive breast cancer probably reflects the indirect effect of frequently coexpressed hormone receptors and adjuvant endocrine therapy.

Abstract OT1-1-11: The B-YOND study: A phase II three-arm randomized trial of the combination of tamoxifen plus goserelin acetate with BYL719 or buparlisib (BKM120) in premenopausal patients with hormone receptor-positive/HER2-negative locally advanced or metastatic breast

Abstract Background: The incidence of breast cancer (BC) has been rapidly increasing in many countries in the last decades. In contrast to Western countries, approximately 50% of BC patients in Eastern countries are premenopausal, with hormone receptor-positive (HR+) disease, or luminal subtype by molecular classification. Benefit from the most recent advances in endocrine treatments for patients with HR+ BC is currently confined to postmenopausal women. For premenopausal metastatic BC (MBC) tamoxifen and/or ovary ablation/suppression remain the recommended first-line therapies for metastatic disease through category 2 evidence, calling for further clinical investigations. In the context of growing evidence of the role of PI3K/AKT/mTOR pathway inhibition in enhancing and extending the benefit of endocrine therapies in HR+ MBC pre-clinical and clinical models, the B-YOND study is a phase II, three-arm randomized trial aimed at exploring the PI3K inhibitors buparlisib and BYL719 in combination with tamoxifen in the context of ovarian suppression in premenopausal patients with MBC. Design & Objectives: Premenopausal women with HR+/HER2-negative locally advanced or MBC who a) are newly diagnosed or b) recurred during or after adjuvant treatment with tamoxifen monotherapy, and received no endocrine treatment in the metastatic setting, will be randomized to receive tamoxifen plus goserelin (control arm, Arm 3) or the same combination with buparlisib (Arm 2) or BYL719 (Arm 1) until progression. Stratification based on previous treatment with tamoxifen and presence of liver and/or lung metastasis will be applied. Primary objective is the efficacy comparison of Arm 1 vs Arm 3 and Arm 2 vs Arm 3 in terms of 9-month progression free survival (PFS) rate. Secondary objectives include additional efficacy evaluations (median PFS, overall response rate, clinical benefit), safety and tolerability, quality of life, and pharmacokinetics. Statistical Methods: Based on previous reports, the estimated proportion of patients who are alive without progression at 9 months is 50% for the control arm (P0=0.50), and we hypothesize that this proportion is 75% for both experimental arms (P1=0.75). Using a two-sided 0.05 level of significance for both the comparisons [Arm 1 vs Arm 3; Arm 2 vs Arm 3], with an overall type-I error not greater than 0.10, with 80% power, the estimated patient numbers needed is 58 patients for each arm. With an assumption that approximately 10% patients will be lost to follow-up, a total of 192 patients will need to be randomized to the three treatment arms in 1:1:1 ratio. Accrual This study will be open for recruitment in Taiwan, South Korea, China, Hong Kong and Thailand and potentially extended to additional Eastern countries. Enrollment started in May 2014 and will last for an estimated period of 18 months. Citation Format: Yen-Shen Lu, Yeon Hee Park, Zhimin Shao, Roberta Valenti. The B-YOND study: A phase II three-arm randomized trial of the combination of tamoxifen plus goserelin acetate with BYL719 or buparlisib (BKM120) in premenopausal patients with hormone receptor-positive/HER2-negative locally advanced or metastatic breast [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr OT1-1-11.

Abstract PD6-6: Estrogen receptor (ESR1) mutations confer resistance to hormone therapy using a common mechanism

Abstract Background: The idea that somatic estrogen receptor gene (ESR1) mutations could play an important role in the evolution of hormone-responsive breast cancers was proposed by us with our original identification of two ESR1 mutations at residues K303 and Y537. Technical issues with ESR1 mutation detection and the resulting paucity of reports in tumors led many to assume that ESR1 mutations were not there. However, with recent Next Generation Sequencing of metastatic tumors, mutation of ESR1 is now an accepted certainty. ESR1 mutant allele frequencies vary over a wide dynamic range, and are usually a minority population within tumors. Therefore how does a minor subclonal tumor population drive resistance in metastatic tumors? Methods: MCF-7 cells expressing endogeous wild-type ER was transduced with ESR1 mutants K303R, Y537N, Y537S, and D538G lentivirus and stable clones selected. ER transcriptional assays and growth in soft agar were performed. Digital drop (dd) PCR and primer extension snp detection were used to ascertain mutant:WT ESR1 allele frequency in cell lines, 200 primary tumors from patients treated with tamoxifen monotherapy, and 20 metastatic breast tumors. Results: Mutant ER constitutive transcriptional activity was fully antagonized by the antiestrogens tamoxifen or fulvestrant in MCF-7 stable transfectants. In contrast, soft agar growth of all ESR1 mutant-expressing cells was unexpectedly and completely resistant to the growth inhibitory effects of tamoxifen, although mutant-expressing cells were a minority subpopulation in the stable clones. Therefore, in cells with WT ER co-expression, the mutant resistant phenotype dominates. We found that phosphorylation of IGF1Rß; was constitutively increased in all ESR1-mutant expressing cells. Treatment with a specific IGF1Rß inhibitor in combination with tamoxifen drastically restored hormone sensitivity in cells expressing the ESR1 mutations. These results suggest a convergence in resistance mechanisms between the K303R and Y537 ESR1 mutation hot spots. We are exploring whether the dominant mutant ESR1 resistant phenotype occurs via activation of paracrine mediators, and have identified altered IGF-1 and interleukin 6 signaling in mutant-expressing cells. Mutation detection in a retrospective cohort of primary and metastatic breast tumors is ongoing and will be presented. Conclusions: We hypothesize that the selection of dominant-acting ESR1 mutations in tumors is a key event in breast cancer progression, potentially due to the selective pressure of antiestrogens. The dominant-resistant phenotype of ESR1 mutants in a majority WT background supports the subclonal evolution of ESR1 mutations in breast cancer recurrence. A common resistance mechanism (like consitutive IGF1Rß activation) should enable biologic targeting of ESR1 mutation-positive metastatic patients a feasible clinical goal. Support: NIH/NCI R01 CA72038 and CPRIT RP1210732 to SAWF. Citation Format: Luca Gelsomino, Guowei Gu, Yassine Rechoum, Sebastiano Ando', Suzanne AW Fuqua. Estrogen receptor (ESR1) mutations confer resistance to hormone therapy using a common mechanism [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD6-6.

Abstract S1-02: Prognostic effects of gene mutation in estrogen receptor positive breast cancer

Abstract Background: Relationships between recurrent somatic mutations and outcome in estrogen receptor positive (ER+) breast cancer has not been extensively studied as the original discovery efforts were from either heterogeneously treated patients or follow up was too brief. Targeted massively parallel sequencing (MPS) analysis was therefore conducted on DNA extracted from archived formalin-fixed breast primaries from a cohort of over 600 patents from British Columbia treated with five years of adjuvant tamoxifen monotherapy and followed for over 10 years (Nielsen et al CCR 16:5222, 2010). Methods: Genes were selected for targeted sequencing by meta-analysis of five large-scale breast cancer sequencing studies and manual review of breast cancer literature. In total 83 genes were identified and 3286 probes were designed to tile across all known exons. Minimum starting input DNA was 50ng (mean=189.1ng). Illumina sequencing libraries were constructed, indexed, pooled, and enriched for target sequences by hybrid-capture followed by paired-end 100bp reads. The Genome Modeling System was used to perform single-tumor somatic variant prediction. Variant calls were filtered to include only targeted regions and exclude variants with global mutant allele frequencies greater than 0.1% in 1000 genomes or NHLBI exome datasets. Kaplan-Meier analysis and multivariable analysis (clinical features and intrinsic subtype by qPCR) was performed for breast-cancer-specific and relapse free survival. Results: A total of 638 samples met minimum quality controls of 80% targeted space covered at 20X or greater. On average each sample had 332M of aligned bases and a mean coverage of 134.3X. In total 7,159 variants were identified including 3,696 missense, 494 nonsense, and 1,047 frameshift insertions or deletions. Preliminary results indicate significant associations between mutation status and improved survival for PIK3CA, ARID1B, ERBB3, MAP3K1 and GATA3 or worse survival for PTEN, DDR1, TP53 and JAK2. Five Y537N/C, two E380Q and 5 potentially novel ligand-binding-domain mutations were identified in ESR1. Such mutations were recently reported to be associated with resistance to hormone therapy but were discovered here in as much as 1.9% of pre-treatment samples. Analysis will be presented regarding the use of relapse events to differentiate passenger from driver mutations. Conclusion. Multiple recurrently mutated genes have both positive and negative associations with prognosis in tamoxifen montherapy treated breast cancer populations. Associations with poor outcome suggest that PTEN, DDR1, and JAK2 are high priorities for pharmacological interventions. Table 1: Mutations and survival in ER+ breast cancerGeneP-ValueHazard RatioPTEN0.0022.11PIK3CA0.0070.69DDR10.0082.41ARID1B0.010.57ERBB30.020.36MAP3K10.010.5TP530.041.48GATA30.040.44JAK20.052.1 Citation Format: Obi L Griffith, Malachi Griffith, Jingqin Luo, Jasreet Hundall, Christopher A Miller, David E Larson, Robert Fulton, Richard K Wilson, Shuzhen Liu, Samuel Leung, Torsten O Nielsen, Elaine R Mardis, Matthew J Ellis. Prognostic effects of gene mutation in estrogen receptor positive breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr S1-02.

Tamoxifen Monotherapy in the Treatment of Retroperitoneal Fibrosis

Objective: To evaluate the therapeutic effect of tamoxifen monotherapy in patients with retroperitoneal fibrosis (RPF). Patients and Methods: From 2007 on, 31 patients with idiopathic RPF were treated with tamoxifen monotherapy. Follow-up investigations included magnetic resonance imaging, laboratory measurements, registration of side effects and changes or removal of ureteral stents. Data were stored in the Else Kröner-Fresenius Registry of Retroperitoneal Fibrosis. Results: 25 men and 6 women with a mean age of 56.6 years were treated with tamoxifen monotherapy. Mean duration of treatment was 13.3 months, mean follow-up 26.8 months. A total of 44 renal units were affected by hydronephrosis and covered by DJ stents. Radiological regression of fibrosis was detected in 22 cases (71.0%); removal of ureteral stents was possible in 27/44 renal units (61.4%) and 17/29 patients (58.6%), respectively. Most patients showed only mild or no side effects of therapy. In 7 cases (22.3%) tamoxifen therapy had to be abandoned because of severe side effects, progression of fibrosis or persistent intolerance. Conclusions: Tamoxifen is an alternative in the medical treatment of RPF, especially if patients want to avoid glucocorticoids. The potential of regression of fibrosis seems to be slightly inferior and the relapse rate is higher compared to steroids, but the rate of successful DJ removals is comparable.

Loss of B‐cell translocation gene 2 expression in estrogen receptor‐positive breast cancer predicts tamoxifen resistance

B-cell translocation gene 2 (BTG2), a gene suppressed in a subset of aggressive breast cancer, is repressed by estrogen. BTG2 inhibits the expression of HER ligands and promotes AKT activation, which plays an essential role in the tamoxifen resistance of estrogen receptor (ER)-positive breast cancer. To determine if BTG2 expression modifies tamoxifen efficacy, a cohort of 60 patients treated with adjuvant tamoxifen monotherapy was analyzed. We found that increased BTG2 expression showed better clinical survival and was the only independent prognostic factor for disease-free survival (hazard ratio, 0.691; 95% confidence interval, 0.495–0.963; P = 0.029). Tamoxifen suppressed the human epidermal growth factor receptor 2 (HER2)-Akt signaling in BTG2 expressing ER-positive breast cancer cells with a correlated increase in sensitivity, whereas BTG2 knockdown abrogated this sensitivity. Consistent with this observation, tamoxifen significantly suppressed the growth ratio, tumor weight and Ki-67 expression in BTG2 expressing breast cancer xenografts in mice. These studies demonstrate that BTG2 is a significant factor in tamoxifen response, acting through modification of AKT activation in ER-positive/HER2-negative breast cancer.

MnSOD rs4880 and XPD rs13181 polymorphisms predict the survival of breast cancer patients treated with adjuvant tamoxifen

The enzyme manganese superoxide dismutase (MnSOD) defends against oxidative stress caused by reactive oxygen species (ROS), whereas Xeroderma pigmentosum group D (XPD) protein is involved in DNA repair. Polymorphisms in these genes have previously been associated with the outcome of breast cancer.Material and methods. Two gene polymorphisms, the MnSOD Val16Ala (rs4880A> G) and the XPD Lys751Gln (rs13181A> C), were analyzed in a cohort of 396 Finnish breast cancer patients by using PCR-RFLP-based methods in a prospective case-control study. The overall survival (OS), breast cancer-specific survival (BCSS), and relapse-free survival (RFS), assessed by using Kaplan-Meier survival analysis and multivariate Cox regression analysis, were evaluated according to the adjuvant treatments and the rs4880 and rs13181 genotypes.Results. In the combined analysis of rs4880 and rs13181 genotypes for patients treated with adjuvant tamoxifen (TAM) an increasing number of low-risk genotypes (rs4880 AA, rs4880 AG, or rs13181 AA) was significantly associated with better RFS, BCSS, and OS (n = 64). In addition, there was improved BCSS and RFS among TAM-treated patients carrying the wild-type rs4880 A allele as compared with the other genotypes (n = 64). The wild-type rs13181 AA genotype was similarly associated with better RFS and BCSS in the TAM-treated population (n = 65).Conclusion. This is the first study to show that the MnSOD rs4880 and XPD rs13181 polymorphisms may influence the outcome of breast cancer patients receiving adjuvant TAM monotherapy. Patients carrying the rs4880 A allele or rs13181 AA genotype may have a reduced ability to scavenge ROS and repair the DNA damage generated by TAM treatment.

Re: Concordance Between CYP2D6 Genotypes Obtained From Tumor-Derived and Germline DNA

The accuracy of CYP2D6 genotyping and its value for the prediction of tamoxifen outcome are a matter of intense debate (1–11). Technical issues related to genotyping of DNA derived from tumor cores and the resultant unprecedented violation of Hardy-Weinberg equilibrium (HWE) (2) has been at the heart of this debate. Rae et al. (12) published new information comparing CYP2D6 genotypes obtained from various tissue sources. They conclude that “based on less than 10% misclassification rate … this could not alter the conclusions of the CYP2D6 BIG1-98 investigation” (12). We applaud Rae and colleagues for obtaining CYP2D6 genotypes within HWE; however, their results and conclusions are irrelevant to our concerns regarding the validity of the BIG 1-98 data (2). That is, Rae et al. did not duplicate the methods of BIG 1-98, where formalin-fixed, paraffin-embedded (FFPE) tumor cores obtained for somatic biomarker studies were used for DNA extraction and CYP2D6 genotypes (obtained after upward of 60 polymerase chain reaction [PCR] cycles (5)) demonstrated massive deviation from HWE (P = 2.5×10-92). Instead, Rae et al. utilized FFPE cores containing some normal tissue and standard PCR methodology. In his editorial, Berry compared the North Central Cancer Treatment Group (NCCTG) 89-30-52 (13) and BIG 1-98 data (2) (both used tumor cores), and argues that “since both studies had the same HWE status, the Regan study was resoundingly clear in failing to corroborate the Goetz observation” (14). Berry seems to accept the argument that deletion of the CYP2D6 gene in breast tumor tissue is driving the observed departures from HWE, but inappropriately argues that the genomic constitution of the tumor is the relevant aspect of genome biology to consider, even though it is inherited variation in the germline that alters plasma endoxifen concentrations. To address NCCTG 89-30-52 HWE issues (13), CYP2D6 genotyping was repeated at Mayo using DNA from FFPE tissue blocks containing nonmalignant tissue as previously published (1) and submitted to the International Tamoxifen Pharmacogenomics Consortium (ITPC) (15). The CYP2D6*4 genotype met HWE (P = .28) and the CYP2D6 genotype was statistically significantly associated with the risk of recurrence, both within NCCTG 89-30-52 and in postmenopausal ITPC patients receiving tamoxifen monotherapy for five years at 20mg/day (15). To address Berry’s concerns that the ITPC results were “ad hoc,” a secondary analysis in the prospective ABCSG 8 clinical trial (using similar eligibility criteria as ITPC) demonstrated that CYP2D6 genotype was statistically significantly associated with the risk of recurrence or death (8). Medical practice and the evidence supporting it must pass accepted scientific standards. Data that cannot pass minimal standards for quality cannot be used to test hypotheses critically related to patient care. It is unquestionably true that the oncology community generally focuses on the tumor genome to direct treatment decisions. But because tamoxifen is metabolized in the liver, it is the germline genome that is relevant for considering its metabolism. Using tumor genome data to classify individuals with respect to their ability to metabolize tamoxifen is scientifically, medically, and practically inappropriate when an unacceptably high proportion of individuals will be misclassified with respect to their ability to metabolize tamoxifen. Does HWE matter? Assuredly, yes, from any rational point of view.

Re: CYP2D6 Genotyping and the Use of Tamoxifen in Breast Cancer

Donald Berry in his recent editorial (1) regarding the role of CYP2D6 and tamoxifen highlights several controversies that exist in the clinical and biomarker field for women with breast cancer. The International Tamoxifen Pharmacogenomics Consortium (ITPC) was formed with the intent to aggregate, curate, and analyze the data available in published breast cancer studies with the hope of answering the question, “Should CYP2D6 genotyping guide the use of tamoxifen in breast cancer?” The ITPC analyzed nearly 5000 patients regarding the association between CYP2D6 and clinical outcomes (2). These data demonstrated that CYP2D6 genotype was associated with the risk of recurrence or death in those patients who received tamoxifen monotherapy for 5 years but not in patients who received different doses or duration of tamoxifen or those who received chemotherapy along with tamoxifen. We are in agreement with Berry that further research is required to understand these complex relationships, and our intent has always been that others will be able to use the ITPC dataset to understand the data as fully as possible in conjunction with their own research efforts. The complete dataset of genotypes and clinical variables, the analysis code, and the full analyses are transparently available at PharmGKB (http://www.pharmgkb.org). Although we disagree with Berry about his conclusion in answering the all-important question of whether CYP2D6-guided genotype therapy is appropriate, we invite other researchers and practitioners to examine the data from their own perspectives and engage in ongoing efforts to use these data to the benefit of patients with breast cancer.

A relative ordering-based predictor for tamoxifen-treated estrogen receptor-positive breast cancer patients: multi-laboratory cohort validation

Current predictors for estrogen receptor-positive (ER-positive) breast cancer patients receiving tamoxifen are often invalid in inter-laboratory validation. We aim to develop a robust predictor based on the relative ordering of expression measurement (ROE) in gene pairs. Using a large integrated dataset of 420 normal controls and 1,129 ER-positive breast tumor samples, we identified the gene pairs with stable ROEs in normal control and significantly reversed ROEs in ER-positive tumor. Using these gene pairs, we characterized each sample of a cohort of 292 ER-positive patients who received tamoxifen monotherapy for 5 years and then identified relapse risk-associated gene pairs. We extracted a gene pair subset that resulted in the largest positive and negative predictive values for predicting 10-year relapse-free survival (RFS) using a genetic algorithm. A predictor was developed based on the gene pair subset and was validated in 2 large multi-laboratory cohorts (N = 250 and 248, respectively) of ER-positive patients who received 5-year tamoxifen alone. In the first validation cohort, the patients predicted to be tamoxifen sensitive had a 10-year RFS of 91 % (95 % confidence interval [CI] 85-97 %) with an absolute risk reduction of 34 % (95 % CI 17-51 %). The patients predicted to be tamoxifen insensitive had a significantly higher relapse risk than the patients predicted to be tamoxifen sensitive (hazard ratio = 4.99, 95 % CI 2.45-10.17, P = 9.13 × 10(-7)). Similar performance was achieved for the second validation cohort. The predictor performed well in both node-negative and node-positive subsets and added significant predictive power to the clinical parameters. In contrast, 2 previously proposed predictors did not achieve significantly better performances than the baselines of the validation cohorts. In summary, the proposed predictor can accurately and robustly predict tamoxifen sensitivity of ER-positive breast cancer patients and identified patients with a high probability of 10-year RFS following tamoxifen monotherapy.

CYP2D6 Genotyping and the Use of Tamoxifen in Breast Cancer

Donald Berry in his recent editorial (1) regarding the role of CYP2D6 and tamoxifen highlights several controversies that exist in the clinical and biomarker field for women with breast cancer. The International Tamoxifen Pharmacogenomics Consortium (ITPC) was formed with the intent to aggregate, curate, and analyze the data available in published breast cancer studies with the hope of answering the question, “Should CYP2D6 genotyping guide the use of tamoxifen in breast cancer?” The ITPC analyzed nearly 5000 patients regarding the association between CYP2D6 and clinical outcomes (2). These data demonstrated that CYP2D6 genotype was associated with the risk of recurrence or death in those patients who received tamoxifen monotherapy for 5 years but not in patients who received different doses or duration of tamoxifen or those who received chemotherapy along with tamoxifen. We are in agreement with Berry that further research is required to understand these complex relationships, and our intent has always been that others will be able to use the ITPC dataset to understand the data as fully as possible in conjunction with their own research efforts. The complete dataset of genotypes and clinical variables, the analysis code, and the full analyses are transparently available at PharmGKB (http://www.pharmgkb. org). Although we disagree with Berry about his conclusion in answering the allimportant question of whether CYP2D6guided genotype therapy is appropriate, we invite other researchers and practitioners to examine the data from their own perspectives and engage in ongoing efforts to use these data to the benefit of patients with breast cancer.

Abstract 4349: Genome-wide kinase and phosphatase siRNA screen identifies clinically relevant targets in IGF1R signaling-mediated antiestrogen resistance in breast cancer.

Abstract Antiestrogen resistance of breast cancer has been linked to increased growth factor receptor signaling such as invoked by the insulin-like growth factor 1 (IGF-1) receptor (IGF1R). We have previously shown that increased IGF1R signaling by ectopic IGF1R overexpression in MCF7 cells (MCF7-IGF1R) directly drives an estrogen receptor (ER)-independent cell proliferation in MCF7 cells that leads to antiestrogen resistance. To identify components of the downstream IGF1R signaling pathways that mediate antiestrogen resistance, we have performed a human genome-wide kinase and phosphatase siRNA screen under tamoxifen resistance condition (1 μM 4-OH-tamoxifen, 1 nM estradiol, and 100 ng/ml IGF-1) in MCF7-IGF1R cells. We identified 174 kinases and phosphatases that increased or decreased tamoxifen resistance. Of these, 14 significantly correlated with the time to disease progression of metastatic breast cancer patients treated with first-line tamoxifen monotherapy, including ADRA1B, BMPR1B, CDK10, CDK5, EIF2AK1 and MAP2K5. Low expression of these six genes in patients correlated with short time to disease progression and silencing of these genes in MCF7-IGF1R cells further increased proliferation under tamoxifen resistance condition. Low expression of the other eight genes in patients correlated with longer time the time to disease progression and silencing of these genes in MCF7-IGF1R cells decreased proliferation under tamoxifen resistance condition Therefore, these latter genes may represent novel drug targets that may be useful to restore tamoxifen sensitivity in breast cancer. Knockdown of two of these genes suppressed IGF1R-mediated effects and restored the anti-proliferative effect of tamoxifen in a dose dependent manner, suggesting synthetic lethality between the knockdowns and tamoxifen. Further study of one of these genes revealed that stable knockdown inhibits proliferation and three-dimensional growth under tamoxifen resistance condition, accompanied with an increase in G1/G0 arrest and mild induction of apoptosis. These effects are independent of PI3K/AKT and MAPK/ERK signaling pathways, and suggest that an as yet unknown pathway plays a decisive role in tamoxifen resistance induced by IGF1R signaling. Citation Format: John H. Meerman, Yinghui Zhang, Maurice P. Jansen, Ramakrishnaiah Siddapa, Sreenivasa Ramaiahgari, Leo Price, Erik Danen, Els M. Berns, John Foekens, Bob van de Water. Genome-wide kinase and phosphatase siRNA screen identifies clinically relevant targets in IGF1R signaling-mediated antiestrogen resistance in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4349. doi:10.1158/1538-7445.AM2013-4349

Ki-67 evaluation at the hottest spot predicts clinical outcome of patients with hormone receptor-positive/HER2-negative breast cancer treated with adjuvant tamoxifen monotherapy

The clinicopathological importance of Ki-67 in breast cancers has been intensely studied; however, there have been few systematic large studies of patients treated with predefined adjuvant therapy. Further, Ki-67 evaluation methodology differed among studies, which prevents Ki-67 from being used for clinical practice. We performed a large systematic study using routinely processed tissues and compared various scoring methods. Representative slides of archival tissue blocks of 442 consecutive invasive breast cancers from women treated with adjuvant tamoxifen monotherapy and having a long follow-up period were subjected to immunohistochemistry using anti-Ki-67 monoclonal antibody, Mib-1. Both the average score across the section and the score at the hottest spot were assessed. Ki-67 evaluated at the hottest spot, not the average score across the section, independently predicted poor clinical outcomes of patients with hormone receptor-positive/HER2-negative cancer. Ki-67 was not a predictor of clinical outcome in patients with triple-negative breast cancer. Overall, high Ki-67 level significantly correlated with classic unfavorable clinicopathological factors, correlating negatively with the status of estrogen receptor (ER)-α and progesterone receptor (PR), and positively with HER2 status and grade. ER-β status positively correlated with the Ki-67 level. Ki-67 evaluation at the hottest spot was superior to that determined by average score across the section as a predictor of outcome in patients with hormone receptor-positive/HER2-negative breast cancers treated with endocrine monotherapy. The different result obtained in patients with triple-negative carcinomas needs to be further investigated.

Gene Expression Signatures That Predict Outcome of Tamoxifen-Treated Estrogen Receptor-Positive, High-Risk, Primary Breast Cancer Patients: A DBCG Study

BackgroundTamoxifen significantly improves outcome for estrogen receptor-positive (ER+) breast cancer, but the 15-year recurrence rate remains 30%. The aim of this study was to identify gene profiles that accurately predicted the outcome of ER+ breast cancer patients who received adjuvant Tamoxifen mono-therapy.Methodology/Principal FindingsPost-menopausal breast cancer patients diagnosed no later than 2002, being ER+ as defined by >1% IHC staining and having a frozen tumor sample with >50% tumor content were included. Tumor samples from 108 patients treated with adjuvant Tamoxifen were analyzed for the expression of 59 genes using quantitative-PCR. End-point was clinically verified recurrence to distant organs or ipsilateral breast. Gene profiles were identified using a model building procedure based on conditional logistic regression and leave-one-out cross-validation, followed by a non-parametric bootstrap (1000x re-sampling). The optimal profiles were further examined in 5 previously-reported datasets containing similar patient populations that were either treated with Tamoxifen or left untreated (n = 623). Three gene signatures were identified, the strongest being a 2-gene combination of BCL2-CDKN1A, exhibiting an accuracy of 75% for prediction of outcome. Independent examination using 4 previously-reported microarray datasets of Tamoxifen-treated patient samples (n = 503) confirmed the potential of BCL2-CDKN1A. The predictive value was further determined by comparing the ability of the genes to predict recurrence in an additional, previously-published, cohort consisting of Tamoxifen-treated (n = 58, p = 0.015) and untreated patients (n = 62, p = 0.25).Conclusions/SignificanceA novel gene expression signature predictive of outcome of Tamoxifen-treated patients was identified. The validation suggests that BCL2-CDKN1A exhibit promising predictive potential.

Side Effects Associated with Ultrarapid Cytochrome P450 2D6 Genotype among Women with Early Stage Breast Cancer Treated with Tamoxifen

The side effects of tamoxifen, a drug widely used for the treatment and the prevention of recurrence in patients with estrogen receptor positive breast cancers (ER+), have been reported in clinical trials, but to date no information is available on their possible association with an increased enzymatic activity of CYP2D6 (ultra-metabolizers, UMs). The aim of this study was therefore to evaluate the association between the presence of multiple functional CYP2D6 alleles and the occurrence of side effects. 61 women with ER+ breast cancer receiving tamoxifen monotherapy were investigated in order to assess the relationships between CYP2D6 UM phenotype and side effects. Genotyping of 16 CYP2D6 polymorphisms was performed using a new DNA microarray technology. A highly significant difference was detected (41.2% of difference, 95% CI 6 - 61%, Fisher's exact test, p = 0.030) between the numbers of Ultrarapid Metabolizer patients (UM; high activity) with two or more adverse drug reactions to tamoxifen (7/9; 77.8%), compared to the number of Extensive Metabolizers (EM; normal activity), Intermediate Metabolizers (IM; reduced activity), and Poor Metabolizers (PM; no activity) with at least two side effects (19/52, 36.5%). A similar difference was also observed comparing the two groups (UM vs EM-IM-PM) for the number of side effects (median and inter quartile range, IQR: AM/EM/IM 1, IQR 0-2 vs. ULTRA 2, IQR 2-4; Mann-Whitney p = 0.005). Our results suggest a new association between CYP2D6 gene duplication and side effects to tamoxifen, indicating a possible role of CYP2D6 in their occurrence.

Abstract S4-4: Independent validation of Genomic Grade in the BIG 1-98 study

Abstract Background: We have reported that a 97-gene signature, the Genomic Grade Index (GGI) can separate histological grade (HG) 2 breast tumors into low vs high GGI tumors with different outcomes. We have also developed a quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) tool including 6 reporter and 3 control genes that can reliably evaluate Genomic Grade (GG) in paraffin embedded tumors. Methods: We evaluated the qRT-PCR GG in women treated with either tamoxifen or letrozole monotherapy in the Breast International Group (BIG) 1–98 study with 8.1 year median follow-up. The association between continuous GG and distant recurrence-free interval (DRFI) was evaluated in Cox regression models stratified for the 2 vs 4 arm randomization option, chemotherapy and hormonotherapy use, with and without adjustment for clinicopathological characteristics. Estrogen, progesterone receptor (ER, PR), Ki67 and HG were centrally reviewed. The clinicopathological model included age and log2 tumor size, ER and PR as continuous variables, nodal status (N0 vs 1–3 vs >3), HG (1 vs 2 vs 3) and HER2 (negative vs positive). Similar analyses were performed for log2 Ki67 as continuous variable. Added prognostic value was assessed using the likelihood ratio statistic. Results: We obtained GG in 883 (62%) of 1428 samples. Non evaluable results were due to pre-analytical issues or technical failure. Among the 883 patients, 521 (59%) had N0 disease, 435 (49%) were treated with tamoxifen and 84 (10%) had distant recurrences. The GG classified 502 patients with HG2 tumors as either GG1 (N = 202, 40%), equivocal (N = 220, 44%) or GG3 (N = 80, 16%). In univariate analysis, increasing GG and Ki67 were significantly associated with lower DRFI (Table). When the 773 N0/1-3 patients were analyzed based on HG, the Kaplan-Meier estimate for 10-year DRFI was 98% (95% confidence intervals 96–100%) for the 123 HG1, 92% (88–95%) for the 456 HG2 and 84% (78–90%) for the 194 HG3 patients. In the N0/1-3 population, 10 year DRFI based on GG was 95% (96–100%) for the 290 GG1, 92% (89–95%) for the 309 GG equivocal and 84% (77–90%) for the 178 GG3 patients. Interestingly, when the 456 HG2 and N0/1-3 patients were analyzed based on GG, the 10-year DRFI was 95% (92–100%) for the 185 HG2/GG1 patients, 92% (88–96%) for the 202 HG2/GG equivocal patients, 86% (76–96%) for the 69 HG2/GG3 patients. In all patients either GG or Ki67 as continuous variable significantly improved the clinicopathological model in predicting distant recurrence (Table). Conclusion: Either Genomic Grade or centrally reviewed Ki67 provides independent prognostic information for risk of distant recurrence beyond clinicopathological characteristics in patients treated with endocrine therapy. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr S4-4.

Abstract P4-09-04: Gene expression profile that predict outcome of Tamoxifen-treated estrogen receptor-positive, high-risk, primary breast cancer patients: a DBCG study.

Abstract Background: Tamoxifen significantly improves outcome for estrogen receptor-positive (ER+) breast cancer, but the 15-year recurrence rate remains 30%. The aim of this study was to identify gene profiles that accurately predicted the outcome of ER+ breast cancer patients who received adjuvant Tamoxifen mono-therapy. Methods: Post-menopausal breast cancer patients diagnosed no later than 2002, being ER+ as defined by >1% IHC staining and having a frozen tumor sample with >50% tumor content were included. Tumor samples from 108 patients treated with adjuvant Tamoxifen were analyzed for the expression of 59 genes using quantitative-PCR. End-point was clinically verified recurrence to distant organs or ipsilateral breast. Gene profiles were identified using a model building procedure based on conditional logistic regression and leave-one-out cross-validation, followed by a non-parametric boostrap (1000x re-sampling). The optimal profiles were further examined in 5 previously-reported datasets containing similar patient populations that were either treated with Tamoxifen or left untreated (N = 623). Results: Three gene profiles were identified, the strongest being a 2-gene combination of BCL2-CDKN1A exhibiting an accuracy of 75% for prediction of outcome. Independent examination using 4 previously-reported microarray datasets of Tamoxifen-treated patient samples (N = 503) confirmed the potential of BCL2-CDKN1A (average accuracy: 68%, range: 53%–85%). The predictive value was further determined by comparing the ability of the genes to predict recurrence in an additional, previously-published, cohort consisting of Tamoxifen-treated (n = 58, p = 0.015) and untreated patients (n = 62, p = 0.25). Conclusion: A novel gene expression profile predictive of outcome of Tamoxifen-treated patients was identified. The validation suggests that BCL2-CDKN1A exhibit strong predictive potential. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-09-04.

Long-term safety and efficacy of a tamoxifen-based treatment strategy for idiopathic retroperitoneal fibrosis

BackgroundTamoxifen may be a viable treatment option for idiopathic retroperitoneal fibrosis (iRPF) but data are limited and its long-term safety and efficacy is unclear. We describe the long-term course and outcomes in a large group of patients with iRPF treated with tamoxifen monotherapy. MethodsThis is a single-center prospective, observational study of 55 patients with iRPF treated with tamoxifen for 2years from April 1998 through April 2011. Measurements included clinical improvement, laboratory parameters and follow-up computed tomographic (CT) scanning. Treatment success was the composite endpoint of clinical improvement, mass regression and definite resolution of ureteral obstruction. ResultsForty-seven (85%) patients reported substantial resolution of symptoms after median treatment duration of 3.0weeks (IQR 1.4–4.8weeks). Repeated CT scanning showed mass regression in 39 (71%) patients at 4months and 47 (85%) patients at 8months of follow-up, respectively. Nineteen (34.5%) patients did not meet the composite endpoint of treatment success, 56% of whom responded satisfactorily to second-line immunosuppressive treatment. Recurrence-free survival in patients with treatment success after post-treatment follow-up of 21months (IQR 9.0–35.0months) was 68%. Tamoxifen was well tolerated. Pulmonary embolism occurred in 2 patients receiving tamoxifen and in one patient receiving second-line treatment. ConclusionTamoxifen is a safe and viable therapeutic option in the treatment of iRPF.

Global microRNA expression profiling of high-risk ER+ breast cancers from patients receiving adjuvant tamoxifen mono-therapy: a DBCG study.

PurposeDespite the benefits of estrogen receptor (ER)-targeted endocrine therapies in breast cancer, many tumors develop resistance. MicroRNAs (miRNAs) have been suggested as promising biomarkers and we here evaluated whether a miRNA profile could be identified, sub-grouping ER+ breast cancer patients treated with adjuvant Tamoxifen with regards to probability of recurrence.Experimental DesignGlobal miRNA analysis was performed on 152 ER+ primary tumors from high-risk breast cancer patients with an initial discovery set of 52 patients, followed by two independent test sets (N = 60 and N = 40). All patients had received adjuvant Tamoxifen as mono-therapy (median clinical follow-up: 4.6 years) and half had developed distant recurrence (median time-to-recurrence: 3.5 years). MiRNA expression was examined by unsupervised hierarchical clustering and supervised analysis, including clinical parameters as co-variables.ResultsThe discovery set identified 10 highly significant miRNAs that discriminated between the patient samples according to outcome. However, the subsequent two independent test sets did not confirm the predictive potential of these miRNAs. A significant correlation was identified between miR-7 and the tumor grade. Investigation of the microRNAs with the most variable expression between patients in different runs yielded a list of 31 microRNAs, eight of which are associated with stem cell characteristics.ConclusionsBased on the large sample size, our data strongly suggests that there is no single miRNA profile predictive of outcome following adjuvant Tamoxifen treatment in a broad cohort of ER+ breast cancer patients. We identified a sub-group of Tamoxifen-treated breast cancer patients with miRNA-expressing tumors associated with cancer stem cell characteristics.